In excitable cells, a small Ca2+ influx through the surface membrane may activate intracellular Ca2+ release channels called ryanodine receptors (RyRs) on the endoplasmic or sarcoplasmic reticulum (SR). The resulting RyR-mediated Ca2+ release regulates many cellular processes like contraction, secretion, synaptic transmission, fertilization, nuclear pore regulation and transcription. Here, the case in point is cardiac muscle. In these cells, surface depolarization activates an L-type Ca2+ channel generating a small Ca2+ flux which activates type-2 ryanodine receptors (RyR2) on the sarcoplasmic reticulum (SR). Activation of multiple RyR2 channels at discrete sites on the SR generates localized Ca2+ release events called sparks. In cells, sparks are the elemental unit of RyR2-mediated Ca2+ release. Recruitment and summation of many sparks generates the global Ca2+ release phenomena that drive cardiac contractility. The local control mechanisms that govern the RyR2-mediated spark are poorly understood. One of these mechanisms is local RyR2 Ca2+ activation, often referred to as Ca2+-induced Ca2+ release (CICR). CICR is an intuitively a self-reinforcing process whose "explosive" positive feedback (i.e., released Ca2+ should trigger further release) should ultimately empty the SR Ca2+ store. This does not happen in cells. Instead, CICR is precisely controlled indicating that some negative-feedback mechanism(s) must exist to counter the inherent positive feedback of CICR. Two cytosolic mechanisms, Ca2+-dependent inactivation and Ca2+-dependent adaptation, have been proposed to be the stabilizing negative feedback. It has also been proposed that the needed negative control may arise from RyR2 regulation by local [Ca2+] changes inside the SR. Delineating mechanisms that govern RyR2 local Ca2+ control is clearly essential to understanding the origin of the Ca2+ spark. This is our focus here and the following hypothesis will be tested. Single RyR2 channels are driven by multiple forms of cytosolic Ca2+ feedback (e.g., feed through, neighbor-induced & paired pulse facilitation). This is controlled/countered by a combination of lumenal negative control mechanisms (e.g., Ca2+-flux reduction, direct & indirect Ca2+ deactivation) to ultimately define the spatiotemporal nature of the Ca2+ spark. The specific aims are: Specific Aim #1: Define the cytosolic local Ca2+ positive feedback that drives the function of single RyR2 Ca2+ release channels. Specific Aim #2: Define lumenal Ca2+ negative control mechanisms that govern operation of single RyR2 Ca2+ release channels and ultimately the spatiotemporal nature of the Ca2+ spark. [unreadable] [unreadable]